连接蛋白43通过蛋白激酶A介导丝氨酸373调控脓毒症急性肺损伤肺泡Ⅱ型上皮细胞屏障功能的研究  

Connexin 43 regulates the barrier function of alveolar typeⅡepithelial cells in sepsis-induced acute lung injury through serine 373 mediated by protein kinase A

作　　者：赵希伟 周佳伟 刘凯 侯林义[3] 张文凯[3] Zhao Xiwei;Zhou Jiawei;Liu Kai;Hou Linyi;Zhang Wenkai(Department of Cardiothoracic Surgery,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China;Department of Hepatobiliary Surgery,Hubei General Hospital,Wuhan 430060,China;Department of Critical Care Medicine,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第二医院心胸外科,太原030001 [2]湖北省人民医院肝胆外科,武汉430060 [3]山西医科大学第二医院重症医学科,太原030001

出　　处：《中华危重症医学杂志（电子版）》2021年第5期355-361,共7页Chinese Journal of Critical Care Medicine:Electronic Edition

基　　金：山西省太原市科技项目(12016905)。

摘　　要：目的观察脓毒症急性肺损伤肺泡Ⅱ型上皮细胞中连接蛋白43(Cx43)表达水平与肺泡气血屏障通透性的关系,并探讨Cx43及蛋白激酶A(PKA)信号通路在脓毒症急性肺损伤中的作用。方法将A549细胞分为对照组、脓毒症组[脂多糖(LPS)组]、LPS+PKA抑制剂组(LPS+H89组)和8-Bromo-cAMP组(PKA激活剂组)。LPS组和LPS+H89组加入LPS 1μg/mL,PKA激活剂组加入8-Bromo-cAMP 10μmol/L,均处理24 h;其中,LPS+H89组予H8910μmol/L预处理1 h后弃去。采用Western-blotting法检测各组细胞磷酸化Cx43(p-Cx43)、PKA及磷酸化PKA(p-PKA)的蛋白表达水平,并用Transwell板进行单层细胞培养,用酶标仪测定A549单层细胞的通透性。结果4组细胞p-Cx43、PKA和p-PKA蛋白表达水平及细胞通透性比较,差异均有统计学意义(F=8.961、249.729、7526.430、3661.755,P均<0.05)。进一步两两比较发现,LPS组和PKA激活剂组p-Cx43和PKA蛋白表达水平及细胞通透性均较对照组显著升高,LPS组、LPS+H89组和PKA激活剂组p-PKA蛋白表达水平均较对照组显著升高;LPS+H89组p-Cx43和PKA蛋白表达水平及细胞通透性均较LPS组降低,p-PKA则较LPS组有所升高;PKA激活剂组较LPS组和LPS+H89组细胞通透性均显著增大(P均<0.05)。结论Cx43通过PKA介导丝氨酸373调控在脓毒症引起的急性肺损伤中起着关键作用,其可增加肺泡Ⅱ型上皮细胞的通透性,破坏肺泡屏障功能及增加缝隙连接细胞间通讯。Objective To observe the relationship between connexin 43(Cx43)of alveolar typeⅡepithelial cells and the permeability of alveolar air blood barrier in sepsis-induced acute lung injury,and to explore the role of Cx43 and protein kinase A(PKA)signaling pathway in sepsis-induced acute lung injury.Methods A549 cells were divided into a control group,a sepsis group[lipopolysaccharide(LPS)group],a LPS+PKA inhibitor group(LPS+H89 group)and a 8-Bromo-cAMP group(PKA activator group).Both the LPS group and LPS+H89 group were treated with LPS 1μg/mL,while the PKA activator group was treated with 8-Bromo-cAMP 10μmol/L,all for 24 h.In addition,the LPS+H89 group was pretreated with H8910μmol/L for 1 h.The protein expression levels of phosphorylated Cx43(p-Cx43),PKA and phosphorylated PKA(p-PKA)in each group were determined by the Western-blotting assay.Monolayer cell cultures were performed with Transwell plates,and the permeability of A549 monolayers was determined by a microplate reader.Results The p-Cx43,PKA and p-PKA protein expression levels and the cell permeability were significantly different among the four groups(F=8.961,249.729,7526.430,3661.755;all P<0.05).Further pairwise comparisons revealed that compared with the control group,the p-Cx43 and PKA protein expression levels and the cell permeability significantly increased in the LPS and PKA activator groups,and the p-PKA protein expression levels significantly increased in the LPS,LPS+H89 and PKA activator groups(all P<0.05).The p-Cx43 and PKA protein expression levels and the cell permeability reduced,while the p-PKA protein expression levels increased in the LPS+H89 group compared with the LPS group(all P<0.05).The cell permeability in the PKA activator group significantly increased compared with the LPS and LPS+H89 groups(both P<0.05).Conclusion Cx43 plays a key role in sepsis-induced acute lung injury through regulating serine 373 mediated by PKA,which can increase permeability of alveolar typeⅡepithelial cells,disrupt alveolar barrier function and incre

关 键 词：脓毒症 急性肺损伤 蛋白激酶A 连接蛋白43 肺泡Ⅱ型上皮细胞 

分 类 号：R459.7[医药卫生—急诊医学] R563[医药卫生—治疗学]